Modified cea nucleic acid and expression vectors

ABSTRACT

The present invention relates to a nucleic acid encoding a polypeptide and the use of the nucleic acid or polypeptide in preventing and/or treating cancer. The invention relates to improved vectors for the insertion and expression of foreign genes encoding tumor antigens for use in immunotherapeutic treatment of cancer. One such foreign DNA sequence is modified CEA nucleic acid.

FIELD OF THE INVENTION

The present invention relates to a nucleic acid encoding a polypeptideand the use of the nucleic acid or polypeptide in preventing and/ortreating cancer. In particular, the invention relates to improvedvectors for the insertion and expression of foreign genes encoding tumorantigens for use in immunotherapeutic treatment of cancer.

BACKGROUND OF THE INVENTION

There has been tremendous increase in last few years in the developmentof cancer vaccines with Tumour-associated antigens (TAAs) due to thegreat advances in identification of molecules based on the expressionprofiling on primary tumours and normal cells with the help of severaltechniques such as high density microarray, SEREX, immunohistochemistry(IHC), RT-PCR, in-situ hybridization (ISH) and laser capture microscopy(Rosenberg, Immunity, 1999; Sgroi et al, 1999, Schena et al, 1995,Offringa et al, 2000). The TAAs are antigens expressed or over-expressedby tumour cells and could be specific to one or several tumours forexample CEA antigen is expressed in colorectal, breast and lung cancers.Sgroi et al (1999) identified several genes differentially expressed ininvasive and metastatic carcinoma cells with combined use of lasercapture microdissection and cDNA microarrays. Several delivery systemslike DNA or viruses could be used for therapeutic vaccination againsthuman cancers (Bonnet et al, 2000) and can elicit immune responses andalso break immune tolerance against TAAs. Tumour cells can be renderedmore immunogenic by inserting transgenes encoding T cell co-stimulatorymolecules such as B7.1 or cytokines IFNgamma, IL2, GM-CSF etc.Co-expression of a TAA and a cytokine or a co-stimulatory molecule candevelop effective therapeutic vaccine (Hodge et al, 95, Bronte et al,1995, Chamberlain et al, 1996).

There is a need in the art for reagents and methodologies useful instimulating an immune response to prevent or treat cancers. The presentinventions provides such reagents and methodologies which overcome manyof the difficulties encountered by others in attempting to treat cancerssuch as cancer. In particular, the present invention provides a novelcoding sequence for CEA. This nucleotide sequence, CEA(6D)-1,2, includessequence modifications that eliminate the expression of truncated formsof CEA as expressed from expression vectors. Such a modified sequence isdesired by those of skill in the art to improve expression andimmunization protocols for CEA.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic target for administrationto a patient to prevent and/or treat cancer. In particular, theimmunogenic target is a CEA tumor antigen (“TA”) and/or anangiogenesis-associated antigen (“AA”). In one embodiment, theimmungenic target is encoded by a modified CEA nucleotide sequence(CEA(6D)-1,2) that improves CEA expression in transfected cells. Incertain embodiments, the TA and/or AA are administered to a patient as anucleic acid contained within a plasmid or other delivery vector, suchas a recombinant virus. The TA and/or AA may also be administered incombination with an immune stimulator, such as a co-stimulatory moleculeor adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A. Illustration of plasmid p3′H6MCEA comprising the CEA codingsequence with the 6D modification under the control of partial H6promoter. B. Illustration of plasmid pSE1544.9 (pUC18-mCEA repeat 1).

FIG. 2. Illustration of plasmid pSEI 616.44 (pUC18-mCEA-modified repeat1).

FIG. 3. Illustration of plasmid pSE1658.15 (p3′H6MCEA-modified repeat1).

FIG. 4. Illustration of plasmid pBSmCEA.

FIG. 5. Illustration of plasmid pSE1686.1 (pUC18 mCEA modified repeat 2.

FIG. 6. Illustration of plasmid pSE1696.1 (pUC18 mCEA modified repeat 2.

FIG. 7. Illustration of plasmid p3′H6modMCEA-1st&2nd repeats.

FIG. 8. Illustration of plasmid pNVQH6MCEA(6D1st&2^(nd)).

FIG. 9A-D. Comparison of nucleotide sequence of CAP(6D) and CAP(6D)-1,2.Differences between the sequences are underlined.

FIG. 10. PCR analysis to confirm the presence of CAP(6D)-1,2 in NYVACDNA.

FIG. 11. Immunoblot illustrating the lack of truncated CEA in cellsexpressing CAP(6D)-1,2.

DETAILED DESCRIPTION

The present invention provides reagents and methodologies useful fortreating and/or preventing cancer. All references cited within thisapplication are incorporated by reference.

In one embodiment, the present invention relates to the induction orenhancement of an immune response against one or more tumor antigens(“TA”) to prevent and/or treat cancer. In certain embodiments, one ormore TAs may be combined. In preferred embodiments, the immune responseresults from expression of a TA in a host cell following administrationof a nucleic acid vector encoding the tumor antigen or the tumor antigenitself in the form of a peptide or polypeptide, for example.

As used herein, an “antigen” is a molecule (such as a polypeptide) or aportion thereof that produces an immune response in a host to whom theantigen has been administered. The immune response may include theproduction of antibodies that bind to at least one epitope of theantigen and/or the generation of a cellular immune response againstcells expressing an epitope of the antigen. The response may be anenhancement of a current immune response by, for example, causingincreased antibody production, production of antibodies with increasedaffinity for the antigen, or an increased cellular response (i.e.,increased T cells). An antigen that produces an immune response mayalternatively be referred to as being immunogenic or as an immunogen. Indescribing the present invention, a TA may be referred to as an“immunogenic target”.

TA includes both tumor-associated antigens (TAAs) and tumor-specificantigens (TSAs), where a cancerous cell is the source of the antigen. ATAA is an antigen that is expressed on the surface of a tumor cell inhigher amounts than is observed on normal cells or an antigen that isexpressed on normal cells during fetal development. A TSA is an antigenthat is unique to tumor cells and is not expressed on normal cells. TAfurther includes TAAs or TSAs, antigenic fragments thereof, and modifiedversions that retain their antigenicity.

TAs are typically classified into five categories according to theirexpression pattern, function, or genetic origin: cancer-testis (CT)antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens(i.e., Melan A/MART-1, tyrosinase, gp100); mutational antigens (i.e.,MUM-1, p53, CDK-4); overexpressed ‘self’ antigens (i.e., HER-2/neu,p53); and, viral antigens (i.e., HPV, EBV). For the purposes ofpracticing the present invention, a suitable TA is any TA that inducesor enhances an anti-tumor immune response in a host to whom the TA hasbeen administered. Suitable TAs include, for example, gp100 (Cox et al.,Science, 264:716-719 (1994)), MART-1/Melan A (Kawakami et al., J. Exp.Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al., J. Exp. Med.,186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol.,24:759-764 (1994); WO 200175117; WO 200175016; WO 200175007), NY-ESO-1(WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J.Immunol., 130:1467-1472 (1983)), MAGE family antigens (i.e., MAGE-1,2,3,4,6,12, 51; Van der Bruggen et al., Science, 254:1643-1647 (1991);U.S. Pat. No. 6,235,525; CN 1319611), BAGE family antigens (Boel et al.,Immunity, 2:167-175 (1995)), GAGE family antigens (i.e., GAGE-1,2; Vanden Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S. Pat. No.6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler et at.,Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526),N-acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med.,183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950(1995)), β-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)),MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)),cyclin dependent kinase-4 (CDK4) (Wolfe) et al., Science, 269:1281-1284(1995)), p21-ras (Fossum et at., Int. J. Cancer, 56:40-45 (1994)),BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald etal., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu(erb-B1; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermalgrowth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat,29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl.Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087;5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and,EP 784483); carcinoma-associated mutated mucins (i.e., MUC-1 geneproducts; Jerome et al., J. Immunol., 151:1654-1662 (1993)); EBNA geneproducts of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys,13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing etal., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA;Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membraneantigen (PSMA; Israeli, et al., Cancer Res., 54:1807-1811 (1994));idiotypic epitopes or antigens, for example, immunoglobulin idiotypes orT cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787(1994)); KSA (U.S. Pat. No. 5,348,887), kinesin 2 (Dietz, et al. BiochemBiophys Res Commun 2000 Sep. 7 ;275(3):731-8), HIP-55, TGFβ-1anti-apoptotic factor (Toomey, et al. Br J Biomed Sci2001;58(3):177-83), tumor protein D52 (Bryne J. A., et al., Genomics,35:523-532 (1996)), HIFT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75,NY-BR-85, NY-BR-87, NY-BR-96 (Scanlan, M. Serologic and BioinformaticApproaches to the Identification of Human Tumor Antigens, in CancerVaccines 2000, Cancer Research Institute, New York, N.Y.), AAC2-1, orAAC2-2, including “wild-type” (i.e., normally encoded by the genome,naturally-occurring), modified, and mutated versions as well as otherfragments and derivatives thereof. Any of these TAs may be utilizedalone or in combination with one another in a co-immunization protocol.

In certain cases, it may be beneficial to co-immunize patients with bothTA and other antigens, such as angiogenesis-associated antigens (“AA”).An AA is an immunogenic molecule (i.e., peptide, polypeptide) associatedwith cells involved in the induction and/or continued development ofblood vessels. For example, an AA may be expressed on an endothelialcell (“EC”), which is a primary structural component of blood vessels.Where the cancer is cancer, it is preferred that that the AA be foundwithin or near blood vessels that supply a tumor. Immunization of apatient against an AA preferably results in an anti-AA immune responsewhereby angiogenic processes that occur near or within tumors areprevented and/or inhibited.

Exemplary AAs include, for example, vascular endothelial growth factor(i.e., VEGF; Bernardi, et al. J. Urol., 2001, 166(4): 1275-9; Starnes,et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23), the VEGFreceptor (i.e., VEGF-R, flk-1/KDR; Starnes, et al. J. Thorac.Cardiovasc. Surg., 2001, 122(3): 518-23), EPH receptors (i.e., EPHA2;Gerety, et al. 1999, Cell, 4: 403-414), epidermal growth factor receptor(i.e., EGFR; Ciardeillo, et al. Clin. Cancer Res., 2001, 7(10):2958-70), basic fibroblast growth factor (i.e., bFGF; Davidson, et al.Clin. Exp. Metastasis 2000, 18(6): 501-7; Poon, et al. Am J. Surg.,2001, 182(3):298-304), platelet-derived cell growth factor (i.e.,PDGF-B), platelet-derived endothelial cell growth factor (PD-ECGF; Hong,et al. J. Mol. Med., 2001, 8(2):141-8), transforming growth factors(i.e., TGF-α; Hong, et al. J. Mol. Med., 2001, 8(2):141-8), endoglin(Balza, et al. Int. J. Cancer, 2001, 94: 579-585), Id proteins (Benezra,R. Trends Cardiovasc. Med., 2001, 11(6):237-41), proteases such as uPA,uPAR, and matrix metalloproteinases (MMP-2, MMP-9; Djonov, et al. J.Pathol., 2001, 195(2):147-55), nitric oxide synthase (Am. J.Ophthalmol., 2001, 132(4):551-6), aminopeptidase (Rouslhati, E. NatureCancer, 2: 84-90, 2002), thrombospondins (i.e., TSP-1, TSP-2; Alvarez,et al. Gynecol. Oncol., 2001, 82(2):273-8; Seki, et al. Int. J. Oncol.,2001, 19(2):305-10), k-ras (Zhang, et al. Cancer Res., 2001,61(16):6050-4), Wnt (Zhang, et al. Cancer Res., 2001, 61(16):6050-4),cyclin-dependent kinases (CDKs; Drug Resist. Updat. 2000, 3(2):83-88),microtubules (Timar, et al. 2001. Path. Oncol. Res., 7(2): 85-94), heatshock proteins (i.e., HSP90 (Timar, supra)), heparin-binding factors(i.e., heparinase; Gohji, et al. Int. J. Cancer, 2001, 95(5):295-301),synthases (i.e., ATP synthase, thymidilate synthase), collagenreceptors, integrins (i.e., ανβ3, ανβ5, α1β1, α2β1, α5β1), the surfaceproteolglycan NG2, AAC2-1 (SEQ ID NO.:1), or AAC2-2 (SEQ ID NO.:2),among others, including “wild-type” (i.e., normally encoded by thegenome, naturally-occurring), modified, mutated versions as well asother fragments and derivatives thereof. Any of these targets may besuitable in practicing the present invention, either alone or incombination with one another or with other agents.

In certain embodiments, a nucleic acid molecule encoding an immunogenictarget is utilized. The nucleic acid molecule may comprise or consist ofa nucleotide sequence encoding one or more immunogenic targets, orfragments or derivatives thereof, such as that contained in a DNA insertin an ATCC Deposit. The term “nucleic acid sequence” or “nucleic acidmolecule” refers to a DNA or RNA sequence. The term encompassesmolecules formed from any of the known base analogs of DNA and RNA suchas, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine, among others.

An isolated nucleic acid molecule is one that: (1) is separated from atleast about 50 percent of proteins, lipids, carbohydrates, or othermaterials with which it is naturally found when total nucleic acid isisolated from the source cells; (2) is not be linked to all or a portionof a polynucleotide to which the nucleic acid molecule is linked innature; (3) is operably linked to a polynucleotide which it is notlinked to in nature; and/or, (4) does not occur in nature as part of alarger polynucleotide sequence. Preferably, the isolated nucleic acidmolecule of the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use. As used herein, the term “naturally occurring” or “native”or “naturally found” when used in connection with biological materialssuch as nucleic acid molecules, polypeptides, host cells, and the like,refers to materials which are found in nature and are not manipulated byman. Similarly, “non-naturally occurring” or “non-native” as used hereinrefers to a material that is not found in nature or that has beenstructurally modified or synthesized by man.

The identity of two or more nucleic acid or polypeptide molecules isdetermined by comparing the sequences. As known in the art, “identity”means the degree of sequence relatedness between nucleic acid moleculesor polypeptides as determined by the match between the units making upthe molecules (i.e., nucleotides or amino acid residues). Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., an algorithm). Identitybetween nucleic acid sequences may also be determined by the ability ofthe related sequence to hybridize to the nucleic acid sequence orisolated nucleic acid molecule. In defining such sequences, the term“highly stringent conditions” and “moderately stringent conditions”refer to procedures that permit hybridization of nucleic acid strandswhose sequences are complementary, and to exclude hybridization ofsignificantly mismatched nucleic acids. Examples of “highly stringentconditions” for hybridization and washing are 0.015 M sodium chloride,0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. (see, for example,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual(2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson el al., NucleicAcid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited)). Theterm “moderately stringent conditions” refers to conditions under whicha DNA duplex with a greater degree of base pair mismatching than couldoccur under “highly stringent conditions” is able to form. Exemplarymoderately stringent conditions are 0.015 M sodium chloride, 0.0015 Msodium citrate at 50-65° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 20% formamide at 37-50° C. By way of example, moderatelystringent conditions of 50° C. in 0.015 M sodium ion will allow about a21% mismatch. During hybridization, other agents may be included in thehybridization and washing buffers for the purpose of reducingnon-specific and/or background hybridization. Examples are 0.1% bovineserum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate,0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll, Denhardt'ssolution, sonicated salmon sperm DNA (or another non-complementary DNA),and dextran sulfate, although other suitable agents can also be used.The concentration and types of these additives can be changed withoutsubstantially affecting the stringency of the hybridization conditions.Hybridization experiments are usually carried out at pH 6.8-7.4;

however, at typical ionic strength conditions, the rate of hybridizationis nearly independent of pH.

In preferred embodiments of the present invention, vectors are used totransfer a nucleic acid sequence encoding a polypeptide to a cell. Avector is any molecule used to transfer a nucleic acid sequence to ahost cell. In certain cases, an expression vector is utilized. Anexpression vector is a nucleic acid molecule that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of the transferred nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and splicing, if introns are present.Expression vectors typically comprise one or more flanking sequencesoperably linked to a heterologous nucleic acid sequence encoding apolypeptide. Flanking sequences may be homologous (i.e., from the samespecies and/or strain as the host cell), heterologous (i.e., from aspecies other than the host cell species or strain), hybrid (i.e., acombination of flanking sequences from more than one source), orsynthetic, for example.

A flanking sequence is preferably capable of effecting the replication,transcription and/or translation of the coding sequence and is operablylinked to a coding sequence. As used herein, the term operably linkedrefers to a linkage of polynucleotide elements in a functionalrelationship. For instance, a promoter or enhancer is operably linked toa coding sequence if it affects the transcription of the codingsequence. However, a flanking sequence need not necessarily becontiguous with the coding sequence, so long as it functions correctly.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence may still be considered operably linked to thecoding sequence. Similarly, an enhancer sequence may be located upstreamor downstream from the coding sequence and affect transcription of thesequence.

In certain embodiments, it is preferred that the flanking sequence is atrascriptional regulatory region that drives high-level gene expressionin the target cell. The transcriptional regulatory region may comprise,for example, a promoter, enhancer, silencer, repressor element, orcombinations thereof. The transcriptional regulatory region may beeither constitutive, tissue-specific, cell-type specific (i.e., theregion is drives higher levels of transcription in a one type of tissueor cell as compared to another), or regulatable (i.e., responsive tointeraction with a compound such as tetracycline). The source of atranscriptional regulatory region may be any prokaryotic or eukaryoticorganism, any vertebrate or invertebrate organism, or any plant,provided that the flanking sequence functions in a cell by causingtranscription of a nucleic acid within that cell. A wide variety oftranscriptional regulatory regions may be utilized in practicing thepresent invention.

Suitable transcriptional regulatory regions include the CMV promoter(i.e., the CMV-immediate early promoter); promoters from eukaryoticgenes (i.e., the estrogen-inducible chicken ovalbumin gene, theinterferon genes, the gluco-corticoid-inducible tyrosineaminotransferase gene, and the thymidine kinase gene); and the majorearly and late adenovirus gene promoters; the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304-10): the promoter containedin the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV)(Yamamoto, et al., 1980, Cell 22:787-97); the herpes simplex virusthymidine kinase (HSV-TK) promoter (Wagner el al., 1981, Proc. Natl.Acad. Sci. 78:1444-45); the regulatory sequences of the metallothioninegene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the beta-lactamase promoter (Villa-Kamaroff et al.,1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter(DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Tissue-and/or cell-type specific transcriptional control regions include, forexample, the elastase 1 gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-46; Ornitz elal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986);MacDonald, 1987, Hepatology 7:425-515); the insulin gene control regionwhich is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-22); the immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-58; Adames et al.,1985, Nature 318:533-38; Alexander et al., 1987, Mo!. Cell. Biol.,7:1436-44); the mouse mammary tumor virus control region in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95);the albtimin gene control region in liver (Pinkert el al., 1987, Genesand Devel. 1:268-76); the alpha-feto-protein gene control region inliver (Krumlauf et al:, 1985, Mol. Cell. Biol., 5:1639-48; Hammer etal., 1987, Science 235:53-58); the alpha 1-antitrypsin gene controlregion in liver (Kelsey et al., 1987, Genes and Devel. 1:161-71); thebeta-globin gene control region in myeloid cells (Mogram et al., 1985,Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelinbasic protein gene control region in oligodendrocyte cells in the brain(Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2 genecontrol region in skeletal muscle (Sani, 1985, Nature 314:283-86); thegonadotropic releasing hormone gene control region in the hypothalamus(Mason et al., 1986, Science 234:1372-78), and the tyrosinase promoterin melanoma cells (Hart, I. Semin Oncol 1996 February ;23(1):154-8;Siders, et al. Cancer Gene Ther 1998 September-October ;5(5):281-91),among others. Other suitable promoters are known in the art.

As described above, enhancers may also be suitable flanking sequences.Enhancers are cis-acting elements of DNA, usually about 10-300 by inlength, that act on the promoter to increase transcription. Enhancersare typically orientation- and position-independent, having beenidentified both 5′ and 3′ to controlled coding sequences. Severalenhancer sequences available from mammalian genes are known (i.e.,globin, elastase, albumin, alpha-feto-protein and insulin). Similarly,the SV40 enhancer, the cytomegalovirus early promoter enhancer, thepolyoma enhancer, and adenovirus enhancers are useful with eukaryoticpromoter sequences. While an enhancer may be spliced into the vector ata position 5′ or 3′ to nucleic acid coding sequence, it is typicallylocated at a site 5′ from the promoter. Other suitable enhancers areknown in the art, and would be applicable to the present invention.

While preparing reagents of the present invention, cells may need to betransfected or transformed. Transfection refers to the uptake of foreignor exogenous DNA by a cell, and a cell has been transfected when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art (i.e., Graham et al.,1973, Virology 52:456; Sambrook et al., Molecular Cloning, A LaboratoryManual (Cold Spring Harbor Laboratories, 1989); Davis et al., BasicMethods in Molecular Biology (Elsevier, 1986); and Chu et al., 1981,Gene 13:197). Such techniques can be used to introduce one or moreexogenous DNA moieties into suitable host cells.

In certain embodiments, it is preferred that transfection of a cellresults in transformation of that cell. A cell is transformed when thereis a change in a characteristic of the cell, being transformed when ithas been modified to contain a new nucleic acid. Following transfection,the transfected nucleic acid may recombine with that of the cell byphysically integrating into a chromosome of the cell, may be maintainedtransiently as an episomal element without being replicated, or mayreplicate independently as a plasmid. A cell is stably transformed whenthe nucleic acid is replicated with the division of the cell.

The present invention further provides isolated immunogenic targets inpolypeptide form. A polypeptide is considered isolated where it: (1) hasbeen separated from at least about 50 percent of polynucleotides,lipids, carbohydrates, or other materials with which it is naturallyfound when isolated from the source cell; (2) is not linked (by covalentor noncovalent interaction) to all or a portion of a polypeptide towhich the “isolated polypeptide” is linked in nature; (3) is operablylinked (by covalent or noncovalent interaction) to a polypeptide withwhich it is not linked in nature; or, (4) does not occur in nature.Preferably, the isolated polypeptide is substantially free from anyother contaminating polypeptides or other contaminants that are found inits natural environment that would interfere with its therapeutic,diagnostic, prophylactic or research use.

Immunogenic target polypeptides may be mature polypeptides, as definedherein, and may or may not have an amino terminal methionine residue,depending on the method by which they are prepared. Further contemplatedare related polypeptides such as, for example, fragments, variants(i.e., allelic, splice), orthologs, homologues, and derivatives, forexample, that possess at least one characteristic or activity (i.e.,activity, antigenicity) of the immunogenic target. Also related arepeptides, which refers to a series of contiguous amino acid residueshaving a sequence corresponding to at least a portion of the polypeptidefrom which its sequence is derived. In preferred embodiments, thepeptide comprises about 5-10 amino acids, 10-15 amino acids, 15-20 aminoacids, 20-30 amino acids, or 30-50 amino acids. In a more preferredembodiment, a peptide comprises 9-12 amino acids, suitable forpresentation upon Class I MHC molecules, for example.

A fragment of a nucleic acid or polypeptide comprises a truncation ofthe sequence (i.e., nucleic acid or polypeptide) at the amino terminus(with or without a leader sequence) and/or the carboxy terminus.Fragments may also include variants (i.e., allelic, splice), orthologs,homologues, and other variants having one or more amino acid additionsor substitutions or internal deletions as compared to the parentalsequence. In preferred embodiments, truncations and/or deletionscomprise about 10 amino acids, 20 amino acids, 30 amino acids, 40 aminoacids, 50 amino acids, or more. The polypeptide fragments so producedwill comprise about 10 amino acids, 25 amino acids, 30 amino acids, 40amino acids, 50 amino acids, 60 amino acids, 70 amino acids, or more.Such polypeptide fragments may optionally comprise an amino terminalmethionine residue. It will be appreciated that such fragments can beused, for example, to generate antibodies or cellular immune responsesto immunogenic target polypeptides.

A variant is a sequence having one or more sequence substitutions,deletions, and/or additions as compared to the subject sequence.Variants may be naturally occurring or artificially constructed. Suchvariants may be prepared from the corresponding nucleic acid molecules.In preferred embodiments, the variants have from 1 to 3, or from 1 to 5,or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, orfrom 1 to 30, or from 1 to 40, to 50, or more than 50 amino acidsubstitutions, insertions, additions and/or deletions.

An allelic variant is one of several possible naturally-occurringalternate forms of a gene occupying a given locus on a chromosome of anorganism or a population of organisms. A splice variant is a polypeptidegenerated from one of several RNA transcript resulting from splicing ofa primary transcript. An ortholog is a similar nucleic acid orpolypeptide sequence from another species. For example, the mouse andhuman versions of an immunogenic target polypeptide may be consideredorthologs of each other. A derivative of a sequence is one that isderived from a parental sequence those sequences having substitutions,additions, deletions, or chemically modified variants. Variants may alsoinclude fusion proteins, which refers to the fusion of one or more firstsequences (such as a peptide) at the amino or carboxy terminus of atleast one other sequence (such as a heterologous peptide).

“Similarity” is a concept related to identity, except that similarityrefers to a measure of relatedness which includes both identical matchesand conservative substitution matches. If two polypeptide sequenceshave, for example, 10/20 identical amino acids, and the remainder areall non-conservative substitutions, then the percent identity andsimilarity would both be 50%. If in the same example, there are fivemore positions where there are conservative substitutions, then thepercent identity remains 50%, but the percent similarity would be 75%(15/20). Therefore, in cases where there are conservative substitutions,the percent similarity between two polypeptides will be higher than thepercent identity between those two polypeptides.

Substitutions may be conservative, or non-conservative, or anycombination thereof. Conservative amino acid modifications to thesequence of a polypeptide (and the corresponding modifications to theencoding nucleotides) may produce polypeptides having functional andchemical characteristics similar to those of a parental polypeptide. Forexample, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a non-native residuesuch that there is little or no effect on the size, polarity, charge,hydrophobicity, or hydrophilicity of the amino acid residue at thatposition and, in particlar, does not result in decreased immunogenicity.Suitable conservative amino acid substitutions are shown in Table 1.

TABLE I Original Exemplary Preferred Residues SubstitutionsSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln GlnAsp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala AlaHis Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Leu NorleucineLeu Norleucine, Ile, Val, Met, Ile Ala, Phe Lys Arg, 1,4 Diamino-butyricArg Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr LeuPro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr TyrTrp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Leu Norleucine

A skilled artisan will be able to determine suitable variants ofpolypeptide using well-known techniques. For identifying suitable areasof the molecule that may be changed without destroying biologicalactivity (i.e., MHC binding, immunogenicity), one skilled in the art maytarget areas not believed to be important for that activity. Forexample, when similar polypeptides with similar activities from the samespecies or from other species are known, one skilled in the art maycompare the amino acid sequence of a polypeptide to such similarpolypeptides. By performing such analyses, one can identify residues andportions of the molecules that are conserved among similar polypeptides.It will be appreciated that changes in areas of the molecule that arenot conserved relative to such similar polypeptides would be less likelyto adversely affect the biological activity and/or structure of apolypeptide. Similarly, the residues required for binding to MHC areknown, and may be modified to improve binding. However, modificationsresulting in decreased binding to MHC will not be appropriate in mostsituations. One skilled in the art would also know that, even inrelatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity. Therefore, even areas that may be important for biologicalactivity or for structure may be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the polypeptide structure.

Other preferred polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites have been alteredcompared to the subject amino acid sequence. In one embodiment,polypeptide variants comprise a greater or a lesser number of N-linkedglycosylation sites than the subject amino acid sequence. An N-linkedglycosylation site is characterized by the sequence Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. To affect O-linked glycosylation ofa polypeptide, one would modify serine and/or threonine residues.

Additional preferred variants include cysteine variants, wherein one ormore cysteine residues are deleted or substituted with another aminoacid (e.g., serine) as compared to the subject amino acid sequence set.Cysteine variants are useful when polypeptides must be refolded into abiologically active conformation such as after the isolation ofinsoluble inclusion bodies. Cysteine variants generally have fewercysteine residues than the native protein, and typically have an evennumber to minimize interactions resulting from unpaired cysteines.

In other embodiments, the isolated polypeptides of the current inventioninclude fusion polypeptide segments that assist in purification of thepolypeptides. Fusions can be made either at the amino terminus or at thecarboxy terminus of the subject polypeptide variant thereof. Fusions maybe direct with no linker or adapter molecule or may be through a linkeror adapter molecule. A linker or adapter molecule may be one or moreamino acid residues, typically from about 20 to about 50 amino acidresidues. A linker or adapter molecule may also be designed with acleavage site for a DNA restriction endonuclease or for a protease toallow for the separation of the fused moieties. It will be appreciatedthat once constructed, the fusion polypeptides can be derivatizedaccording to the methods described herein. Suitable fusion segmentsinclude, among others, metal binding domains (e.g., a poly-histidinesegment), immunoglobulin binding domains (i.e., Protein A, Protein G, Tcell, B cell, Fc receptor, or complement protein antibody-bindingdomains), sugar binding domains (e.g., a maltose binding domain), and/ora “tag” domain (i.e., at least a portion of α-galactosidase, a strep tagpeptide, a T7 tag peptide, a FLAG peptide, or other domains that can bepurified using compounds that bind to the domain, such as monoclonalantibodies). This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification of the sequence of interest polypeptide from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purifiedsequence of interest polypeptide by various means such as using certainpeptidases for cleavage. As described below, fusions may also be madebetween a TA and a co-stimulatory components such as the chemokinesCXC10 (IP-10), CCL7 (MCP-3), or CCL5 (RANTES), for example.

A fusion motif may enhance transport of an immunogenic target to an MHCprocessing compartment, such as the endoplasmic reticulum. Thesesequences, referred to as tranduction or transcytosis sequences, includesequences derived from HIV tat (see Kim et al. 1997 J. Immunol.159:1666), Drosophila antennapedia (see Schutze-Redelmeier et al. 1996J. lmmunol. 157:650), or human period-1 protein (hPER1; in particular.SRRHHCRSKAKRSRHH).

In addition, the polypeptide or variant thereof may be fused to ahomologous polypeptide to form a homodimer or to a heterologouspolypeptide to form a heterodimer. Heterologous peptides andpolypeptides include, but are not limited to: an epitope to allow forthe detection and/or isolation of a fusion polypeptide; a transmembranereceptor protein or a portion thereof, such as an extracellular domainor a transmembrane and intracellular domain; a ligand or a portionthereof which binds to a transmembrane receptor protein; an enzyme orportion thereof which is catalytically active; a polypeptide or peptidewhich promotes oligomerization, such as a leucine zipper domain; apolypeptide or peptide which increases stability, such as animmunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide or variant thereof.

In certain embodiments, it may be advantageous to combine a nucleic acidsequence encoding an immunogenic target, polypeptide, or derivativethereof with one or more co-stimulatory component(s) such as cellsurface proteins, cytokines or chemokines in a composition of thepresent invention. The co-stimulatory component may be included in thecomposition as a polypeptide or as a nucleic acid encoding thepolypeptide, for example. Suitable co-stimulatory molecules include, forinstance, polypeptides that bind members of the CD28 family (i.e., CD28,ICOS; IIutloff, et al. Nature 1999, 397: 263-265; Peach, et al. J ExpMed 1994, 180: 2049-2058) such as the CD28 binding polypeptides B7.1(CD80; Schwartz, 1992; Chen et al, 1992; Ellis, et al. J. Immunol.,156(8): 2700-9) and B7.2 (CD86; Ellis, et al. J. Immunol., 156(8):2700-9); polypeptides which bind members of the integrin family (i.e.,LFA-1 (CD11a/CD18); Sedwick, et al. J. Immunol 1999, 162: 1367-1375;Wülfing, et al. Science 1998, 282: 2266-2269; Lub, et al. Immunol Today1995, 16: 479-483) including members of the ICAM family (i.e., ICAM-1,-2 or -3); polypeptides which bind CD2 family members (i.e., CD2,signalling lymphocyte activation molecule (CDw150 or “SLAM”; Aversa, etal. J. Immunol 1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2 ligand;Davis, et al. Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos,et al. Nature 1998, 395: 462-469); polypeptides which bind heat stableantigen (HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27: 2524-2528);polypeptides which bind to members of the TNF receptor (TNFR) family(i.e., 4-1BB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489),OX40 (CD134; Weinberg, et al. Semin Immunol 1998, 10:471-480; Higgins,et al. J Immunol 1999, 162: 486-493), and CD27 (Lens, et al. SeminImmunol 1998, 10: 491-499)) such as 4-1BBL (4-1BB ligand; Vinay, et al.Semin, Immunol 1998, 10: 481-48; DeBenedette, et al. J Immunol 1997,158: 551-559), TNFR associated factor-1 (TRAF-1; 4-1BB ligand; Saoulli,et al. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998,18: 558-565), TRAF-2 (4-1 BB and OX40 ligand; Saoulli, et al. J Exp Med1998, 187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526,Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB andOX40 ligand; Arch, et al. Mol Cell Biol 1998,18: 558-565; Jang, et al.Biochem Biophys. Res Commun, 1998, 242: 613-620; Kawamata S, et al. JBiol Chem 1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. JImmunol 1998, 161: 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. MolCell Biol 1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273:5808-5814), and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther.,5(3): 163-75). CD154 (CD40 ligand or “CD40L”; Gurunathan, et al. J.Immunol., 1998, 161: 4563-4571; Sine, et al. Hum. Gene Ther., 2001, 12:1091-1102) may also be suitable.

One or more cytokines may also be suitable co-stimulatory components or“adjuvants”, either as polypeptides or being encoded by nucleic acidscontained within the compositions of the present invention (Parmiani, etal. Immunol Lett 2000 Sep. 15 ; 74(1): 41-4; Berzofsky, et al. NatureImmunol. 1: 209-219). Suitable cytokines include, for example,interleukin-2 (IL-2) (Rosenberg, et al. Nature Med. 4: 321-327 (1998)),IL-4, IL-7, IL-12 (reviewed by Pardoll, 1992; Harries, et al. J. GeneMed. 2000 July-August ;2(4):243-9; Rao, et al. J. Immunol. 156:3357-3365 (1996)), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16(Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. CancerRes. Clin. Oncol. 2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al.Blood, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-α), orinterferon-gamma (INF-γ). Other cytokines may also be suitable forpracticing the present invention, as is known in the art.

Chemokines may also be utilized. For example, fusion proteins comprisingCXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor self-antigen have beenshown to induce anti-tumor immunity (Biragyn, et al. Nature Biotech.1999, 17: 253-258). The chemokines CCL3 (MIP-1α) and CCL5 (RANTES)(Boyer, et al. Vaccine, 1999, 17 (Supp. 2): S53-S64) may also be of usein practicing the present invention. Other suitable chemokines are knownin the art.

It is also known in the art that suppressive or negative regulatoryimmune mechanisms may be blocked, resulting in enhanced immuneresponses. For instance, treatment with anti-CTLA-4 (Shrikant, et al.Immunity, 1996, 14: 145-155; Sutmuller, et al. J. Exp. Med., 2001, 194:823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al. J.Immunol., 1999, 163: 184-193), the fusion protein IL13Ra2-Fc (Terabe, etal. Nature Immunol., 2000, 1: 515-520), and combinations thereof (i.e.,anti-CTLA-4 and anti-CD25, Sutmuller, supra) have been shown toupregulate anti-tumor immune responses and would be suitable inpracticing the present invention.

Any of these components may be used alone or in combination with otheragents. For instance, it has been shown that a combination of CD80,ICAM-1 and LFA-3 (“TRICOM”) may potentiate anti-cancer immune responses(Hodge, et al. Cancer Res. 59: 5800-5807 (1999). Other effectivecombinations include, for example, IL-12+GM-CSF (Ahlers, et al. J.Immunol., 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol. 158:4591-4601 (1997)), IL-12+GM-CSF+TNF-α (Ahlers, et al. Int. Immunol. 13:897-908 (2001)), CD80+IL-12 (Fruend, et al. Int. J. Cancer, 85: 508-517(2000); Rao, et al. supra), and CD86+GM-CSF+IL-12 (Iwasaki, supra). Oneof skill in the art would be aware of additional combinations useful incarrying out the present invention.ln addition, the skilled artisanwould be aware of additional reagents or methods that may be used tomodulate such mechanisms. These reagents and methods, as well as othersknown by those of skill in the art, may be utilized in practicing thepresent invention.

Additional strategies for improving the efficiency of nucleic acid-basedimmunization may also be used including, for example, the use ofself-replicating viral replicons (Caley, et al. 1999. Vaccine, 17:3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al.2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000. Mol.Ther., 1: 497-500; Dubensky, supra; Huang, et al. 2001. J. Virol. 75:4947-4951), in viva electroporation (Widera, et al. 2000. J. Immunol.164: 4635-3640), incorporation of CpG stimulatory motifs (Gurunathan, etal. Ann. Rev. Immunol., 2000, 18: 927-974; Leitner, supra), sequencesfor targeting of the endocytic or ubiquitin-processing pathways(Thomson, et al. 1998. J. Virol. 72: 2246-2252; Velders, et al. 2001. J.Immunol. 166: 5366-5373), prime-boost regimens (Gurunathan, supra;Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998.Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292: 69-74), and theuse of mucosal delivery vectors such as Salmonella (Darji, et al. 1997.Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Othermethods are known in the art, some of which are described below.

Chemotherapeutic agents, radiation, anti-angiogenic compounds, or otheragents may also be utilized in treating and/or preventing cancer usingimmunogenic targets (Sebti, et al. Oncogene 2000 Dec. 27;19(56):6566-73). For example, in treating Metastatic breast cancer,useful chemotherapeutic agents include cyclophosphamide, doxorubicin,paclitaxel, docetaxel, navelbine, capecitabine, and mitomycin C, amongothers. Combination chemotherapeutic regimens have also proven effectiveincluding cyclophosphamide+methotrexate+5-fluorouracil;cyclophosphamide+doxorubicin+5-fluorouracil; or,cyclophosphamide+doxorubicin, for example. Other compounds such asprednisone, a taxane, navelbine, mitomycin C, or vinblastine have beenutlized for various reasons. A majority of breast cancer patients haveestrogen-receptor positive (ER+) tumors and in these patients, endocrinetherapy (i.e., tamoxifen) is preferred over chemotherapy. For suchpatients, tamoxifen or, as a second line therapy, progestins(medroxyprogesterone acetate or megestrol acetate) are preferred.Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof suchas letrozole) decrease the availability of estrogen needed to maintaintumor growth and may be used as second or third line endocrine therapyin certain patients.

Other cancers may require different chemotherapeutic regimens. Forexample, metastatic colorectal cancer is typically treated withCamptosar (irinotecan or CPT-11), 5-fluorouracil or leucovorin, alone orin combination with one another. Proteinase and integrin inhibitors suchas as the MMP inhibitors marimastate (British Biotech), COL-3(Collagenex), Neovastat (Aeterna), AG3340 (Agouron), BMS-275291 (BristolMyers Squibb), CGS 27023A (Novartis) or the integrin inhibitors Vitaxin(Medimmune), or MED 1522 (Merck KgaA) may also be suitable for use. Assuch, immunological targeting of immunogenic. targets associated withcolorectal cancer could be performed in combination with a treatmentusing those chemotherapeutic agents. Similarly, chemotherapeutic agentsused to treat other types of cancers are well-known in the art and maybe combined with the immunogenic targets described herein.

Many anti-angiogenic agents are known in the art and would be suitablefor co-administration with the immunogenic target vaccines (see, forexample, Timar, et al. 2001. Pathology Oncol. Res., 7(2): 85-94). Suchagents include, for example, physiological agents such as growth factors(i.e., ANG-2, NK1,2,4 (HGF), transforming growth factor beta (TGF-β));cytokines (i.e., interferons such as IFN-α, -β, -γ, platelet factor 4(PF-4), PR-39), proteases (i.e., cleaved AT-III, collagen XVIII fragment(Endostatin)), HinwKallikrein-d5 plasmin fragment (Angiostatin),prothrombin-F1-2, TSP-1), protease inhibitors (i.e., tissue inhibitor ofmetalloproteases such as TIMP-1, -2, or -3; maspin; plasminogenactivator-inhibitors such as PAI-1; pigment epithelium derived factor(PEDF)), Tumstatin (available through ILEX, Inc.), antibody products(i.e., the collagen-binding antibodies HUIV26, HUI77, XL313; anti-VEGF;anti-integrin (i.e., Vitaxin, (Lxsys))), and glycosidases (i.e.,heparinase-I, -III). “Chemical” or modified physiological agents knownor believed to have anti-angiogenic potential include, for example,vinblastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatinA, rapamycin (Guba, et al. 2002, Nature Med., 8: 128-135), CEP-7055(available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (BayerCorp.), AG3340 (Agouron, Inc.), CGS 27023A (Novartis), tetracylcinederivatives (i.e., COL-3 (Collagenix, Inc.)), Neovastat (Aeterna),BMS-275291 (Bristol-Myers Squibb), low dose 5-FU, low dose methotrexate(MTX), irsofladine, radicicol, cyclosporine, captopril, celecoxib,D45152-sulphated polysaccharide, cationic protein (Protamine), cationicpeptide-VEGF, Suramin (polysulphonated napthyl urea), compounds thatinterfere with the function or production of VEGF (i.e., SU5416 orSU6668 (Sugen), PTK787/ZK22584 (Novartis)), Distamycin A, Angiozyme(ribozyme), isoflavinoids, staurosporine derivatives, genistein,EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid,carboxyamidotriazole, TNP-470, octreotide, 2-methoxyestradiol,aminosterols (i.e., squalamine), glutathione analogues (i.e.,N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptorblocking agents (Nature, 414:933-938, 2001), Rh-Angiostatin,Rh-Endostatin (WO 01/93897), cyclic-RGD peptide, accutin-disintegrin,benzodiazepenes, humanized anti-avb3 Ab, Rh-PAI-2, amiloride,p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab,L-phanylalanin-N-methylamides (i.e., Batimistat, Marimastat), AG3340,and minocycline. Many other suitable agents are known in the art andwould suffice in practicing the present invention.

The present invention may also be utilized in combination with“non-traditional” methods of treating cancer. For example, it hasrecently been demonstrated that administration of certain anaerobicbacteria may assist in slowing tumor growth. In one study, Clostridiumnovyi was modified to eliminate a toxin gene carried on a phage episomeand administered to mice with colorectal tumors (Dang, et al. P.N.A.S.USA, 98(26): 15155-15160, 2001). In combination with chemotherapy, thetreatment was shown to cause tumor necrosis in the animals. The reagentsand methodologies described in this application may be combined withsuch treatment methodologies.

Nucleic acids encoding immunogenic targets may be administered topatients by any of several available techniques. Various viral vectorsthat have been successfully utilized for introducing a nucleic acid to ahost include retrovirus, adenovirus, adeno-associated virus (AAV),herpes virus, and poxvirus, among others. It is understood in the artthat many such viral vectors are available in the art. The vectors ofthe present invention may be constructed using standard recombinanttechniques widely available to one skilled in the art. Such techniquesmay be found in common molecular biology references such as MolecularCloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego,Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis,et al. 1990. Academic Press, San Diego, Calif.).

Preferred retroviral vectors are derivatives of lentivirus as well asderivatives of murine or avian retroviruses. Examples of suitableretroviral vectors include, for example, Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number ofretroviral vectors can incorporate multiple exogenous nucleic acidsequences. As recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided by, for example, helper cell lines encodingretrovirus structural genes. Suitable helper cell lines include ψ2,PA317 and PA12, among others. The vector virions produced using suchcell lines may then be used to infect a tissue cell line, such as NIH3T3 cells, to produce large quantities of chimeric retroviral virions.Retroviral vectors may be administered by traditional methods (i.e.,injection) or by implantation of a “producer cell line” in proximity tothe target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5(3): 343-79; Culver, K., et al., Cold Spring Harb. Symp. Quant. Biol.,59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4(1): 39-69). Theproducer cell line is engineered to produce a viral vector and releasesviral particles in the vicinity of the target cell. A portion of thereleased viral particles contact the target cells and infect thosecells, thus delivering a nucleic acid of the present invention to thetarget cell. Following infection of the target cell, expression of thenucleic acid of the vector occurs.

Adenoviral vectors have proven especially useful for gene transfer intoeukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004):431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (1): 42-51), the studyeukaryotic gene expression (Levrero, M., et al., 1991, Gene, 101 (2):195-202), vaccine development (Graham, F. and Prevec, L., 1992,Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet,L., et al., 1992, Bone Marrow Transplant., 9 (Suppl. 1): 151-2 ; Rich,D., et al., 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routesfor administrating recombinant Ad to different tissues in vivo haveincluded intratracheal instillation (Rosenfeld, M., et al, 1992, Cell,68 (1): 143-55) injection into muscle (Quantin, B., et al., 1992, Proc.Natl. Acad. Sci. U.S.A., 89 (7): 2581-4), peripheral intravenousinjection (Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci.U.S.A., 90 (7): 2812-6) and stereotactic inoculation to brain (Le Gal LaSalle, G., et al., 1993, Science, 259 (5097): 988-90), among others.

Adeno-associated virus (AAV) demonstrates high-level infectivity, broadhost range and specificity in integrating into the host cell genome(Hermonat, P., et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81 (20):6466-70). And Herpes Simplex Virus type-1 (HSV-1) is yet anotherattractive vector system, especially for use in the nervous systembecause of its neurotropic property (Geller, A., et al., 1991, TrendsNeurosci., 14 (10): 428-32; Glorioso, el al., 1995, Mol. Biotechnol., 4(1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).

Poxvirus is another useful expression vector (Smith, et al. 1983, Gene,25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al,1992, Curr. Top. Microbiol. Immunol., 158: 25-38; Moss, et al. 1991.Science, 252: 1662-1667). Poxviruses shown to be useful includevaccinia, NYVAC, avipox, fowlpox, canarypox, ALVAC, and ALVAC(2), amongothers.

NYVAC (vP866) was derived from the Copenhagen vaccine strain of vacciniavirus by deleting six nonessential regions of the genome encoding knownor potential virulence factors (see, for example, U.S. Pat. Nos.5,364,773 and 5,494,807). The deletion loci were also engineered asrecipient loci for the insertion of foreign genes. The deleted regionsare: thymidine kinase gene (TK; J2R); hemorrhagic region (u; B13R+B14R);A type inclusion body region (ATI; A26L); hemagglutinin gene (HA; A56R);host range gene region (C7L-K1L); and, large subunit, ribonucleotidereductase (14L). NYVAC is a genetically engineered vaccinia virus strainthat was generated by the specific deletion of eighteen open readingframes encoding gene products associated with virulence and host range.NYVAC has been show to be useful for expressing TAs (see, for example,U.S. Pat. No. 6,265,189). NYVAC (vP866), vP994, vCP205, vCP1433,placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited withthe ATCC under the terms of the Budapest Treaty, accession numbersVR-2559, VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, andATCC-97914, respectively.

ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are alsosuitable for use in practicing the present invention (see, for example,U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1) except thatALVAC(2) genome comprises the vaccinia E3L and K3L genes under thecontrol of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al.,1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). BothALVAC(1) and ALVAC(2) have been demonstrated to be useful in expressingforeign DNA sequences, such as TAs (Tartaglia et al., 1993 a,b; U.S.Pat. No. 5,833,975). ALVAC was deposited under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, USA, ATCC accessionnumber VR-2547.

Another useful poxvirus vector is TROVAC. TROVAC refers to an attenuatedfowlpox that was a plaque-cloned isolate derived from the FP-1 vaccinestrain of fowlpoxvirus which is licensed for vaccination of I day oldchicks. TROVAC was likewise deposited under the terms of the BudapestTreaty with the ATCC, accession number 2553.

“Non-viral” plasmid vectors may also be suitable in practicing thepresent invention. Preferred plasmid vectors are compatible withbacterial, insect, and/or mammalian host cells. Such vectors include,for example, PCR-II, pCR3, and pcDNA3.1.(Invitrogen, San Diego, Calif.),pBSIl (Stratagene, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.),pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, PaloAlto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT pub. No. WO90/14363) and pFastBacDual (Gibco-BRL, Grand Island, N.Y.) as well asBluescript® plasmid derivatives (a high copy number COLEI -basedphagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloningplasmids designed for cloning Taq-amplified PCR products (e.g., TOPO™ TAcloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad,Calif.). Bacterial vectors may also be used with the current invention.These vectors include, for example, Shigella, Salmonella, Vibriocholerae, Lactobacillus, Bacille guérin (BCG), and Streptococcus (seefor example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO92/21376). Many other non-viral plasmid expression vectors and systemsare known in the art and could be used with the current invention.

Suitable nucleic acid delivery techniques include DNA-ligand complexes,adenovirus-Inland-DNA complexes, direct injection of DNA, CaPO₄precipitation, gene gun techniques, electroporation, and colloidaldispersion systems, among others. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome, which are artificial membrane vesicles usefulas delivery vehicles in vitro and in vivo. RNA, DNA and intact virionscan be encapsulated within the aqueous interior and be delivered tocells in a biologically active form (Fraley, R., et al., 1981, TrendsBiochem. Sci ., 6: 77). The composition of the liposome is usually acombination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

An immunogenic target may also be administered in combination with oneor more adjuvants to boost the immune response. Exemplary adjuvants areshown in Table I below:

TABLE I Types of Immunologic Adjuvants Type of Adjuvant General ExamplesSpecific Examples/References 1 Gel-type Aluminum hydroxide/phosphate(Aggerbeck and Heron, 1995) (“alum adjuvants”) Calcium phosphate(Relyveld, 1986) 2 Microbial Muramyl dipeptide (MDP) (Chedid et al.,1986) Bacterial exotoxins Cholera toxin (CT), E. coli labile toxin(LT)(Freytag and Clements, 1999) Endotoxin-based adjuvantsMonophosphoryl lipid A (MPL) (Ulrich and Myers, 1995) Other bacterialCpG oligonucleotides (Corral and Petray, 2000), BCG sequences (Krieg, etal. Nature, 374: 576). tetanus toxoid (Rice, et al. J. Immunol., 2001,167: 1558-1565) 3 Particulate Biodegradable (Gupta et al., 1998) polymermicrospheres Immunostimulatory complexes (Morein and Bengtsson, 1999)(ISCOMs) Liposomes (Wassef et al., 1994) 4 Oil-emulsion and Freund'sincomplete adjuvant (Jensen et al., 1998) surfactant-based adjuvantsMicrofluidized emulsions MF59 (Ott et al., 1995) SAF (Allison and Byars,1992) (Allison, 1999) Saponins QS-21 (Kensil, 1996) 5 Synthetic Muramylpeptide derivatives Murabutide (Lederer, 1986) Threony-MDP (Allison,1997) Nonionic block copolymers L121 (Allison, 1999) Polyphosphazene(PCPP) (Payne et al., 1995) Synthetic polynucleotides Poly A: U, Poly I:C (Johnson, 1994)

The immunogenic targets of the present invention may also be used togenerate antibodies for use in screening assays or for immunotherapy.Other uses would be apparent to one of skill in the art. The term“antibody” includes antibody fragments, as are known in the art,including Fab, Fab₂, single chain antibodies (Fv for example), humanizedantibodies, chimeric antibodies, human antibodies, produced by severalmethods as are known in the art. Methods of preparing and utilizingvarious types of antibodies are well-known to those of skill in the artand would be suitable in practicing the present invention (see, forexample, Harlow, et al. Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988; Harlow, et al. Using Antibodies: A LaboratoryManual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature,256:495 (1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann etal. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol.,2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988);Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992);Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13(1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos.4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and,5,661,016). The antibodies or derivatives therefrom may also beconjugated to therapeutic moieties such as cytotoxic drugs or toxins, oractive fragments thereof such as diptheria A chain, exotoxin A chain,ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin,among others. Cytotoxic agents may also include radiochemicals.Antibodies and their derivatives may be incorporated into compositionsof the invention for use in vitro or in vivo.

Nucleic acids, proteins, or derivatives thereof representing animmunogenic target may be used in assays to determine the presence of adisease state in a patient, to predict prognosis, or to determine theeffectiveness of a chemotherapeutic or other treatment regimen.Expression profiles, performed as is known in the art, may be used todetermine the relative level of expression of the immunogenic target.The level of expression may then be correlated with base levels todetermine whether a particular disease is present within the patient,the patient's prognosis, or whether a particular treatment regimen iseffective. For example, if the patient is being treated with aparticular chemotherapeutic regimen, an decreased level of expression ofan immunogenic target in the patient's tissues (i.e., in peripheralblood) may indicate the regimen is In decreasing the cancer load in thathost. Similarly, if the level of expresssion is increasing, anothertherapeutic modality may need to be utilized. In one embodiment, nucleicacid probes corresponding to a nucleic acid encoding an immunogenictarget may be attached to a biochip, as is known in the art, for thedetection and quantification of expression in the host.

It is also possible to use nucleic acids, proteins, derivativestherefrom, or antibodies thereto as reagents in drug screening assays.The reagents may be used to ascertain the effect of a drug candidate onthe expression of the immunogenic target in a cell line, or a cell ortissue of a patient. The expression profiling technique may be combinedwith high throughput screening techniques to allow rapid identificationof useful compounds and monitor the effectiveness of treatment with adrug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8(1998)). Drug candidates may be chemical compounds, nucleic acids,proteins, antibodies, or derivatives therefrom, whether naturallyoccurring or synthetically derived. Drug candidates thus identified maybe utilized, among other uses, as pharmaceutical compositions foradministration to patients or for use in further screening assays.

Administration of a composition of the present invention to a host maybe accomplished using any of a variety of techniques known to those ofskill in the art. The composition(s) may be processed in accordance withconventional methods of pharmacy to produce medicinal agents foradministration to patients, including humans and other mammals (i.e., a“pharmaceutical composition”). The pharmaceutical composition ispreferably made in the form of a dosage unit containing a given amountof DNA, viral vector particles, polypeptide or peptide, for example. Asuitable daily dose for a human or other mammal may vary widelydepending on the condition of the patient and other factors, but, onceagain, can be determined using routine methods.

The pharmaceutical composition may be administered orally, parentally,by inhalation spray, rectally, intranodally, or topically in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. The term “pharmaceutically acceptablecarrier” or “physiologically acceptable carrier” as used herein refersto one or more formulation materials suitable for accomplishing orenhancing the delivery of a nucleic acid; polypeptide, or peptide as apharmaceutical composition. A “pharmaceutical composition” is acomposition comprising a therapeutically effective amount of a nucleicacid or polypeptide. The terms “effective amount” and “therapeuticallyeffective amount” each refer to the amount of a nucleic acid orpolypeptide used to induce or enhance an effective immune response. Itis preferred that compositions of the present invention provide for theinduction or enhancement of an anti-tumor immune response in a hostwhich protects the host from the development of a tumor and/or allowsthe host to eliminate an existing tumor from the body.

For oral administration, the pharmaceutical composition may be of any ofseveral forms including, for example, a capsule, a tablet, a suspension,or liquid, among others. Liquids may be administered by injection as acomposition with suitable carriers including saline, dextrose, or water.The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, intrasternal, infusion, or intraperitonealadministration. Suppositories for rectal administration of the drug canbe prepared by mixing the drug with a suitable non-irritating excipientsuch as cocoa butter and polyethylene glycols that are solid at ordinarytemperatures but liquid at the rectal temperature.

The dosage regimen for immunizing a host or otherwise treating adisorder or a disease with a composition of this invention is based on avariety of factors, including the type of disease, the age, weight, sex,medical condition of the patient, the severity of the condition, theroute of administration, and the particular compound employed. Forexample, a poxviral vector may be administered as a compositioncomprising 1×10⁶ infectious particles per dose. Thus, the dosage regimenmay vary widely, but can be determined routinely using standard methods.

A prime-boost regimen may also be utilized (WO 01/30382 A1) in which thetargeted immunogen is initially administered in a priming step in oneform followed by a boosting step in which the targeted immunogen isadministered in another form. The form of the targeted immunogen in thepriming and boosting steps are different. For instance, if the primingstep utilized a nucleic acid, the boost may be administered as apeptide. Simmilarly, where a priming step utilized one type ofrecombinant virus (i.e., ALVAC), the boost step may utilize another typeof virus (i.e., NYVAC). This prime-boost method of administration hasbeen shown to induce strong immunological responses.

While the compositions of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other compositions or agents (i.e., other immunogenictargets, co-stimulatory molecules, adjuvants). When administered as acombination, the individual components can be formulated as separatecompositions administered at the same time or different times, or thecomponents can be combined as a single composition.

Injectable preparations, such as sterile injectable aqueous oroleaginous suspensions, may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Theinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent.Suitable vehicles and solvents that may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution, among others. Forinstance, a viral vector such as a poxvirus may be prepared in 0.4%NaCl. In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose, any bland fixed oil maybe employed, including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

For topical administration, a suitable topical dose of a composition maybe administered one to four, and preferably two or three times daily.The dose may also be administered with intervening days during which nodoes is applied. Suitable compositions may comprise from 0.001% to 10%w/w, for example, from 1% to 2% by weight of the formulation, althoughit may comprise as much as 10% w/w, but preferably not more than 5% w/w,and more preferably from 0.1% to 1% of the formulation. Formulationssuitable for topical administration include liquid or semi-liquidpreparations suitable for penetration through the skin (e.g., liniments,lotions, ointments, creams, or pastes) and drops suitable foradministration to the eye, ear, or nose.

The pharmaceutical compositions may also be prepared in a solid form(including granules, powders or suppositories). The pharmaceuticalcompositions may be subjected to conventional pharmaceutical operationssuch as sterilization and/or may contain conventional adjuvants, such aspreservatives, stabilizers, wetting agents, emulsifiers, buffers etc.Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may also comprise, as innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting sweetening,flavoring, and perfuming agents.

Pharmaceutical compositions comprising a nucleic acid or polypeptide ofthe present invention may take any of several forms and may beadministered by any of several routes. In preferred embodiments, thecompositions are administered via a parenteral route (intradermal,intramuscular or subcutaneous) to induce an immune response in the host.Alternatively, the composition may be administered directly into a lymphnode (intranodal) or tumor mass (i.e., intratumoral administration). Forexample, the dose could be administered subcutaneously at days 0, 7, and14. Suitable methods for immunization using compositions comprising TAsare known in the art, as shown for p53 (Hollstein et al., 1991), p21-ras(Almoguera et al., 1988), HER-2 (Fendly et al., 1990), themelanoma-associated antigens (MAGE-1; MAGE-2) (van der Bruggen et al.,1991), p97 (Hu et al., 1988), and carcinoembryonic antigen (CEA) (Kantoret al., 1993; Fishbein et al., 1992; Kaufman et al., 1991), amongothers.

Preferred embodiments of administratable compositions include, forexample, nucleic acids or polypeptides in liquid preparations such assuspensions, syrups, or elixirs. Preferred injectable preparationsinclude, for example, nucleic acids or polypeptides suitable forparental, subcutaneous, intradermal, intramuscular or intravenousadministration such as sterile suspensions or emulsions. For example, arecombinant poxvirus may be in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose or the like. The composition may also be provided in lyophilizedform for reconstituting, for instance, in isotonic aqueous, salinebuffer. In addition, the compositions can be co-administered orsequentially administered with other antineoplastic, anti-tumor oranti-cancer agents and/or with agents which reduce or alleviate illeffects of antineoplastic, anti-tumor or anti-cancer agents.

A kit comprising a composition of the present invention is alsoprovided. The kit can include a separate container containing a suitablecarrier, diluent or excipient. The kit can also include an additionalanti-cancer, anti-tumor or antineoplastic agent and/or an agent thatreduces or alleviates ill effects of antineoplastic, anti-tumor oranti-cancer agents for co- or sequential-administration. Additionally,the kit can include instructions for mixing or combining ingredientsand/or administration.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

Examples Example 1

A. Modification of mCEA (6D) repeat 1

The presence of truncated forms of CEA in cells following expression ofrecombinant CEA has been documented. This study set forth to generateCEA-encoding nucleic acid sequences that do not result in the expressionof truncated CEA following expression in cells. Generation andexpression of a new CEA-encoding nucleic acid sequence, CAP(6D)-1,2, isdescribed below.

The plasmid p3′H6MCEA was obtained from Virogenetics, Inc. This plasmidcontains the MCEA gene with 6D modification under the control of partialH6 promoter (FIG. 1A). The 912 by Nrul-BamHI fragment from p3′H6MCEA wascloned into pUC18 to form plasmid pSE1544.9 (pUC18-mCEA repeat 1; FIG.1B).

OPC purified Oligos 7524-7526, 7528-7533, 7535-7537, and 7567-7568 werekinased and annealed to create two fragments which were ligated toresult in a 464 by synthetic modified mCEA repeat 1 flanked by AccI andBamHI sites. This synthetic modified repeat 1 fragment was cloned intopSE1544.9 AccI-BamHI to create pSE1616.44 (pUC18-mCEA-modified repeat 1;FIG. 2). The 904 by EcoRV-BamHI fragment of pSE1616.44 was cloned backinto p3′H6MCEA EcoRV-BamHI to form pSE1658.15 (p3′H6MCEA-modified repeat1; FIG. 3).

B. Modification of mCEA(6D) Repeat 2

A synthetic modified repeat 2 fragment was created by using a methodcalled gene splicing by overlap extension (SOE) and cloned intopBluescript-SK+, generating pBSmCEA (FIG. 4). The oligos used for therepeat 2 modification are shown below (section IV, B). The two differentclones (pBS-mCEA-3 and pBS-mCEA-8) contained various point mutations.

The 697 by BamHI-EcoRI fragment of pBS-mCEA-3 was cloned into pUC18BamHI-EcoRI to create pSE1671.8. The 591 by SpeI-Bsu361 fragment of pBSmCEA-8 was cloned into pSE1671.8 SpeI-Bsu361, generating plasmiddesignated pSE1681.1. Two site PCR mutagenesis, using the Quikchangesite directed mutagenesis kit from Stratagene with oligos 7751(GGACGGTAGTAGGTGTATGATGGAGATATAGTTGGGTCGTCTGGGCC) and 7760(CAGAATGAATTATCCGTTGATCACTCC), was performed to correct the tworemaining point mutations pSE1681.1. The corrected clone was designatedpSE1686.1 (pUC18 mCEA modified repeat 2; FIG. 5).

As noted recently, an Alanine codon was absent from 5′ terminus of thesecond repeat in is plasmid p3′H6MCEA which contained CEA. To preservethe consistency of the amino acid sequence of CEA, the Alanine codonpresent in plasmid pSE1686.1 containing the modified second repeat ofCEA was knocked out. This was accomplished using oligos 7802(CGTGACGACGATTACCGTGTATGAGCCACCAAAACCATTCATAAC) and 7803(GTTATGAATGGTTTTGGTGGCTCATACACGGTAATCGTCGTCACG) and the Quikchangesite-directed mutagenesis kit from Stratagene. The resulting plasmid,pSE1696.1 (pUC18 mCEA modified repeat 2; FIG. 6) was confirmed bysequencing.

The 694 by Bsu361-BamHI fragment from pSE1696.1 was cloned intoBsu361-BamHI site of Pse1658.15 to combine modified repeats 1 and 2. Thegenerated plasmid was designated p3′H6modMCEA-1st&2nd repeats (FIG. 7).

C. Construction of ALVAC Donor Plasmid pNVQH6MCEA(6D1st&2nd)

The 2.2 kb Nrul/Xhol fragment from p3′H6modMCEA-1st&2nd repeats wascloned into Nrul/Xhol site of pNVQH6LSP-18, generatingpNVQH6MCEA(6D1st&2^(nd); FIG. 8). The modified CEA sequence(“CAP(6D)-1,2”) contained within pNVQH6MCEA is shown in FIG. 9.

D. Expression of Modified CEA

To test the stability of the CAP(6D)-1,2 sequence upon expression in acell, the gene together with flanking H6 promoter was PCR amplifiedusing pNVQH6MCEA(6D1ST&2ND) as template and two oligos (8034LZ:CTGGCGCGCCTTCTTTATTCTATACTTAAAAAGTG; and 8035LZ:CTGGTACCAGAAAAACTATATCAGAGCAACCCCAAC). The PCR product was then clonedinto an NYVAC TK donor plasmid designated pLZTK1 containing the LacZ andK1L marker genes. This vector was specifically made for the generationof recombinant virus in NYVAC by using blue/white screening method.After in vitro recombination between donor plasmid pLZTK1mCCA(6D1st&2nd)and NYVAC, the foreign CAP(6D)-1,2 sequence and marker genes areintegrated into the NYVAC genome. The plaques containing intermediaterecombinant NYVAC with both LacZ and mCEA appeared blue. Several roundsof plaque purification were then performed. The second recombinationevent kicked out the marker genes resulting in the final white plaquescontaining recombinants with only the CAP(6D)-1,2 sequence but no markergenes (FIG. 10).

The recombinant white plaques and blue plaques were picked forconfirmation of CAP(6D)-1,2 sequence expression. Infection was performedusing the virus from the respective plaques and the cells were harvestedthree days after infection for preparing either cellular DNA or celllysate. For isolation of recombinant NYVAC DNA, DNAzol® reagent(GibcoBRL) was used. PCR (PCR Condition: 95° C. (5 min)→[95° C(30sec)→49° C.(30 sec)→72°C.(1 min)]_0 30 cycles→72° C. (7 min)→4° C.) wasrun to confirm the existence of CAP(6D)-1,2 sequence in the recombinantNYVAC genome. The primers used were 7569LZ (5′ttggatccatggagtctccctcggcc 3′ forward primer) and 7570LZ (5′ttggatccctatatcagagcaacccc 3′ reverse primer), which could amplify thefull length 2106 by CAP(6D)-1,2.

The final recombinant white plaques PRBC-III-2, 3, 6, 8, 9, 10 alldemonstrated the 2.1 kb CAP(6D)-1,2 sequence band in PCR. PRBC-III-N1was a blue plaque with both marker genes and CAP(6D)-1,2 sequence stillin the viral genome and the CAP(6D)-1,2 sequence band was also amplifiedin the PCR. The prominent PCR band amplified from vCP 307 DNA(containing native CEA integrated into the ALVAC genome) was truncatedCEA at 1.2 kb with a very faint full-length CEA band. The cell-onlysample (no viral infection) was used as a negative control and theplasmid pLZTK1MCEA(6D1ST&2ND) was a positive control used in the PCRreaction. The PCR results clearly showed the full-length CAP(6D)-1,2 inthe recombinant viral genome with no other truncated form of CEAvisible. This result indicated that CAP(6D)-1,2 has increased stabilityrelative to the native CEA in the ALVAC genome.

Protein expression was also assayed by immunoblot to confirm the absenceof truncated CEA protein in cells expressing CAP(6D)-1,2 (FIG. 11). Forisolation of cell lysate, cells were first washed with PBS followed bythe addition of Lysis Buffer (Reporter Gene Assay; Boehringer Mannheim)and shaking for 15 minutes. Cell lysate was spun down at 13,000 rpm andthe supernatant was collected for Western blot analysis. Samples wereloaded onto a 10% polyacrylamide gel and run at 125 volts. The proteinwas then transferred to a PVDF filter membrane (Immobilon-P, Millipore).An HRP-linked mouse CEA monoclonal antibody (1:1000; Fitzgerald) wasused to detect the expression of mCEA with the enhancement from achemiluminescence reagent (DNA Thunder™; NEN™ Life Science Products).

All six final CAP(6D)-1,2 recombinant white plaques(PRBC-III-2,3,6,8,9,10) and one intermediate blue plaque (pRBC-III-N1)showed only one CEA band with no other truncated form (FIG. 11). Incontrast, protein from vCP307 plaques (recombinant ALVAC expressingnative CEA) showed a clear truncated CEA product at ˜60 kDa in additionto the full length CEA. Prolonged exposure of the film verified theabsence of any truncated CEA polypeptides in the CAP(6D)-1,2recombinants. CEF was used as the negative control.

In conclusion, the CAP(6D)-1,2 recombinants were generated with the mCEAinstead of the native CEA to prevent the expression of multiple versionsof CEA. CAP(6D)-1,2 expressed from recombinant NYVAC was proveneffective in eliminating truncated version of CEA by both PCR andWestern blot.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1-50. (canceled)
 51. A method for preventing or treating cancercomprising administering to a host an isolated expression vectorcomprising SEQ ID NO.:
 28. 52. The method of claim 51 wherein the vectoris a plasmid or a viral vector.
 53. The method of claim 52 wherein theviral vector is selected from the group consisting of poxvirus,adenovirus, retrovirus, herpesvirus, and adeno-associated virus.
 54. Themethod of claim 53 wherein the viral vector is a poxvirus selected fromthe group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC,ALVAC(2), fowlpox, and TROVAC.
 55. The isolated nucleic acid molecule ofclaim 51 further comprising a nucleic acid sequence encoding aco-stimulatory molecule.
 56. The isolated nucleic acid molecule of claim55 wherein the co-stimulatory molecule is human B7.1.
 57. The isolatednucleic acid molecule of claim 51 further comprising a nucleic acidsequence encoding at least one additional tumor-associated antigen. 58.The isolated nucleic acid molecule of claim 51 further comprising anucleic acid sequence encoding at least one angiogenesis-associatedantigen.
 59. The method of claim 51 wherein the expression vector isadmixed with a pharmaceutically acceptable carrier.
 60. A method forpreventing or treating cancer comprising administering to a host anisolated expression vector comprising a CEA-encoding nucleic acidsequence including at least the sequence set forth by 421-1490 of SEQ IDNO.:
 28. 61. The method of claim 60 wherein the vector is a plasmid or aviral vector.
 62. The method of claim 61 wherein the viral vector isselected from the group consisting of poxvirus, adenovirus, retrovirus,herpesvirus, and adeno-associated virus.
 63. The method of claim 62wherein the viral vector is a poxvirus selected from the groupconsisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2),fowlpox, and TROVAC.
 64. The isolated nucleic acid molecule of claim 60further comprising a nucleic acid sequence encoding a co-stimulatorymolecule.
 65. The isolated nucleic acid molecule of claim 64 wherein theco-stimulatory molecule is human B7.1.
 66. The isolated nucleic acidmolecule of claim 60 further comprising a nucleic acid sequence encodingat least one additional tumor-associated antigen.
 67. The isolatednucleic acid molecule of claim 60 further comprising a nucleic acidsequence encoding at least one angiogenesis-associated antigen.
 68. Themethod of claim 60 wherein the expression vector is admixed with apharmaceutically acceptable carrier.